Mychonastes sp. 246 Suppresses Human Pancreatic Cancer Cell Growth via IGFBP3-PI3K-mTOR Signaling

Previously, we confirmed that Mychonastes sp. 246 methanolic extract (ME) markedly reduced the viability of BxPC-3 human pancreatic cancer cells. However, the underlying mechanism ME remained unclear. Hence, we attempted to elucidate the anticancer effect of ME on BxPC-3 human pancreatic cancer cells. First, we investigated the components of ME and their cytotoxicity in normal cells. Then, we confirmed the G1 phase arrest mediated growth inhibitory effect of ME using a cell counting assay and cell cycle analysis. Moreover, we found that the migration-inhibitory effect of ME using a Transwell migration assay. Through RNA sequencing, Gene Ontology-based network analysis, and western blotting, we explored the intracellular mechanisms of ME in BxPC-3 cells. ME modulated the intracellular energy metabolism-related pathway by altering the mRNA levels of IGFBP3 and PPARGC1A in BxPC-3 cells and reduced PI3K and mTOR phosphorylation by upregulating IGFBP3 and 4E-BP1 expression. Finally, we verified that ME reduced the growth of three-dimensional (3D) pancreatic cancer spheroids. Our study demonstrates that ME suppresses pancreatic cancer proliferation through the IGFBP3-PI3K-mTOR signaling pathway. This is the first study on the anticancer effect of the ME against pancreatic cancer, suggesting therapeutic possibilities and the underlying mechanism of ME action.

cells (hMSCs) were purchased from Lonza (USA) and cultured in StemMACSTM MSC expansion media (Miltenyi Biotec, Germany). Cells (5 × 10 3 cells/well) were seeded in 96-well plates in a medium containing 5% FBS and 1% antibiotics. After 24 h, the medium was replaced with fresh medium containing each ME concentration for 24, 48, and 72 h. At each time point, the number of viable cells was counted under a microscope (Nikon Eclipse TE300; Nikon, Japan).

Cell Cycle Analysis
BxPC-3 cells (8 × 10 5 cells/ml) were seeded into a 100-mm dish and starved in a serum-free medium for 24 h. After treatment with ME for indicated times (12 or 24 h), the cells were fixed with 70% ethanol in 1X PBS at -20°C for 3 days. The collected cells were washed twice with PBS and resuspended in PBS containing 100 μg/ml propidium iodide (PI) solution (Sigma-Aldrich) and 50 μg/ml RNase A (Sigma-Aldrich) for 45 min at room temperature. Cell fluorescence was measured using CytoFlex flow cytometry (Beckman Coulter, USA) and analyzed using FlowJo version 10 (Beckman Coulter).

Transwell Migration Assay
The migration assay was performed using 24-well plate chambers with Transwell inserts with an 8.0-μm pore (BD Bioscience, USA). First, cells (8 × 10 4 cells/ml) were seeded into the upper chamber with serum-free medium. Next, 1, 5, 10 and 20 μM of ME in 0.8 ml medium, including serum, were added to the lower chamber. After 12-24 h, migrated cells were stained with crystal violet (Sigma-Aldrich) for 10 min. After washing the cells with PBS, the non-migrated cells were removed using a sterilized cotton swap. Finally, the migrated cells were observed and counted under a microscope (Nikon Eclipse TE300; Nikon).

RNA Sequencing
Total RNA was isolated and underwent quality assessment with Agilent 2000 bioanalyzer (Agilent Technologies, Netherlands). The library was generated with NEBNext Ultra II Directional RNA-Seq Kit (New England Biolabs, UK). Next, the mRNA was isolated, fragmented and synthesized using the Poly(A) RNA Selection Kit (Lexogen, Austria) according to the manufacturer's instructions. The Illumina indexes 1-12 were used for indexing. The raw sequencing data underwent quality control by FastQC(https://www.bioinformatics.babraham.ac.uk/projects/ fastqc/). HiSeq X 10 (Illumina, USA) was utilized for high-throughput paired-end 100 sequencing, and the adapter and low-quality reads (< Q20) were excluded by FASTX_Trimmer ((http://hannonlab.cshl.edu/ fastx_toolkit/) and BBMap ((https://sourceforge.net/projects/bbmap). For analysis, the reads were mapped to the reference genome via TopHat [23]and the expression levels were calculated based on the Fragments Per Kilobase Million (FPKM) mapped reads from Cufflinks [24]. Quantile normalization was performed via EdgeR (R Development Core Team, 2016) and ExDEGA (E-biogen, Inc., Korea) was used for mining and graphic visualization of the data. A more detailed description of the method is provided in our previous study [4].

Gene Ontology-Based Network Analysis
The biological functions of the regulated genes (changing over 2-fold by ME treatment) were determined through an interaction network using the STRING database (http://string-db.org/). The biological functions of the differentially expressed genes and proteins were analyzed according to Gene Ontology-related interaction networks, including cell death-and proliferation-related signaling. Network generation was modulated based on the obtained expression profiles to produce highly connected networks (Network type, full STRING network; meaning of network edges, evidence; minimum required interaction score, highest confidence).

Three-Dimensional (3D) Spheroid Culture
Cells were seeded in 96-well ultra-low attachment microplates (500 cells/well) and cultured after centrifugation at 200 ×g for 5 min. Spheroids were allowed to form for three days after seeding before ME treatment. On day 3 of treatment, the medium was replaced with a fresh ME-containing medium. Morphological changes in the spheroids were observed for 12 days, and the volume of the spheroids was evaluated using the NIS-Elements imaging software (Nikon).

Statistical Analyses
Prism (GraphPad Software, USA) was used for statistical analyses. All measurements were performed at least in duplicate, and all values are expressed as the mean ± standard error of the mean. The results were processed using analysis of variance with Tukey's test to assess the statistical significance of the differences between groups. Statistical significance was set at p < 0.05.

Components in ME
First, to investigate the components of ME, we analyzed the ME using a high-resolution mass spectrometer and a traditional medicine library. Through the investigation of ME on positive electrospray ionization (ES+) and negative electrospray ionization (ES-) TOF MS, we examined both positively and negatively charged components in the extract. As shown in Fig. 1 and Table 1, β-sitosterol tetra-O-acetyl-β-D-glucopyranoside, stigmasta-3α,5αdiol-3-O-β-D-glucopyranoside tetraacetate, and glycocholic acid were highly abundant in ME. In addition, the response values of β-sitosterol tetra-O-acetyl-β-D-glucopyranoside, component 12 in ES-and 18 in ES+, were measured over 2-fold than stigmasta-3α,5α-diol-3-O-β-D-glycopyranoside tetraacetate and glycocholic acid, which are the next most abundant components in ME. Thus, β-sitosterol tetra-O-acetyl-β-d-glucopyranoside may be a significant component of ME.

ME Treatment Explicitly Suppresses Pancreatic Cancer Cell Growth, Not Normal Cell Growth
Next, to determine the effects of ME on human pancreatic cancer cell proliferation, BxPC-3 and hMSCs were treated with 0, 10, 50, and 100 μg/ml ME for 24, 48, and 72 h. ME treatment inhibited cell proliferation in a dosedependent manner in pancreatic cancer cells; treatment with 50 μg/ml ME for 24 h blocked cell proliferation by
approximately 20% ( Figs. 2A and 2B). In addition, exposure to 100 μg/ml of ME for 24 h inhibited the proliferation of BxPC-3 cells up to 40%. In contrast, hMSCs did not present any significant decrease in cell viability in response to 24 h of ME treatment up to a concentration of 50 μg/ml (Figs. 2C and 2D). A statistically significant decrease was observed in 24 h of 100 μg/ml treatment, and 50, 100 μg/ml in longer treatment conditions, with the highest toxic effects appearing at 72 h of 100 μg/ml treatment, reaching 82.7% in cell viability. Overall, the toxicity of ME in hMSCs revealed to be significantly lower than in BxPC-3 cells even in high-dose conditions, remaining over 80% of viability, above which is considered inconsequentially toxic, according to previous research [26].
Morphological changes in both cell lines were observed under a light microscope after treatment with different ME concentrations (0, 10, 20, 50, and 100 μg/ml). Exposure to 100 μg/ml ME for 72 h did not induce marked morphological changes in BxPC-3 cells and hMSCs (Fig. 2B). However, ME treatment markedly reduced the density of BxPC-3 cells. Fig. 2 shows that ME treatment did not induce distinct morphological changes, such as shrunken or buoyant cells. Since dead or dying cells switch their morphology, including shrinking, swelling, and floating [4,27], we postulated that ME treatment disturbed pancreatic cancer cell growth and did not trigger cancer cell death. Thus, to investigate how ME treatment modulated cancer cell growth, we performed PI staining and flow cytometric analysis of BxPC-3 cells treated with ME (0, 100, and 200 μg/ml) for 12 and 24 h (Fig. 3). ME treatment (200 μg/ml) decreased relative fraction of cells in the G2/M phase 27.2%-19.6% at 12 h and 25.4%-20.5% at 24 h. The proportion of cells in the S phase was reduced by approximately 32.9%-28.8% after 12 h by 200 μg/ml ME. Contrary to the results seen with the G2/M and S phases, ME treatment (200 μg/ml) increased the proportion of cells in the G1 phase from 38.8% to 45.2% at 12 h and from 47.1% to 51.3% at 24 h. Next, we confirmed the regulation of cell cycle-related protein levels by ME treatment (Fig. S1). Treatment with 200 μg/ml ME for 12 h induced the expression of tumor suppressor genes such as p16 and p21, which regulate cell cycle progression. In addition, G1 checkpoint-related protein pRB was upregulated over 2.5-fold by ME treatment in BxPC-3 cells, and expression of cyclin D1 slightly fluctuated. These data suggest that ME triggers G1 phase arrest following S and G2/M phase reduction in BxPC-3 pancreatic cancer cells.

Mychonastes sp. Treatment Inhibits Migration of Pancreatic Cancer Cells
Pancreatic cancer is difficult to detect in its early stages, and most patients have metastasis at the time of diagnosis [28]. Furthermore, pancreatic cancer is highly aggressive with high metastatic potential [29]. Thus, we performed a Transwell migration assay with ME (0, 1, 5, 10, and 20 μg/ml) to determine the migration-inhibitory effect of ME in pancreatic cancer cells for 16 h (Fig. 4). ME treatment at 20 μg/ml did not markedly affect cancer cell viability. However, the cancer cell migration was blocked by up to 50%. These results suggested that ME even at low toxic concentrations can efficiently block cancer cell migration.

ME Treatment Modulates the Gene Expression of Human Pancreatic Cancer Cells
Figs. 3 and 4 show that ME treatment blocked cell cycle progression and migration in pancreatic cancer cells. To explore the intracellular pathway of ME, we analyzed gene expression levels through RNA sequencing of BxPC-3 cells treated with 100 μg/ml ME for 24 h. Among the 25,737 unique genes evaluated, the expression levels of 30 genes were changed by ME treatment, among which 22 were upregulated and 8 were downregulated. Using the ExDEGA program [30], we combined the biological features of genes and categorized the genes that play a crucial

Fig. 4. Migration-inhibitory effect of Mychonastes sp. 246 methanolic extracts (ME) in BxPC-3 pancreatic cancer cells. (A) Migrated cells were examined using a Transwell migration system and visualized after crystal violet staining.
(B) Data represent the mean ± SEM of three independent experiments. *p < 0.05, and **p < 0.01 vs. control cells. role in cancer cell proliferation. The cell proliferation-related genes regulated by 2-fold in ME-treated BxPC-3 cells were assessed using the Multiple Experiment Viewer (MeV) tool and hierarchical cluster analysis (Fig. 5). We confirmed that ME treatment modulated the expression of 22 genes, among which 8 genes were downregulated, while 14 genes were upregulated. Furthermore, ME treatment blocked cellular growth by regulating insulin-like growth factor-binding protein 6 (IGFBP6) (Fig. 5A). In addition, the expression of peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PPARGC1A, also known as PGC-1α) and CREB-regulated transcription coactivator 2 (CRTC2) was modulated in ME-treated BxPC-3 cells.

Mychonastes sp. Treatment Inhibits mTOR Activation via IGFBP3-PI3K Pathway
As shown in Fig. 5, we confirmed that ME treatment of BxPC-3 cells changed the mRNA levels of IGFBP and AMPK and caloric-related pathways. Based on STRING analysis, we investigated the changes in the intracellular energy-related pathway in ME-treated BxPC-3 cells using western blotting. ME treatment at 100 or 200 μg/ml for 24 h of BxPC-3 cells declined the activation of PI3K and mTOR, and not of Akt, IGFBP3, and 4E-BP1 (Fig. 6). ME treatment at 200 μg/ml for 24 h markedly reduced phosphorylation of PI3K by 0.2-fold and mTOR by 0.6-fold. However, ME treatment induced IGFBP3 expression up to 2.6-fold. In addition, phosphorylation of Akt and 4E-

ME Treatment Suppresses the Growth of 3D Spheroidal BxPC-3 Cells
Although tumors in the body grow in a 3D environment, cancer cells in vitro were cultured in a 2D environment. To compensate for this drawback, we evaluated whether ME treatment decreased the volume of 3D spheroidal BxPC-3 cells. On the third day after seeding, the spheroids were treated with each ME concentration (day 0; Fig. 7). The volume of BxPC-3 spheroids increased for 12 days, but treatment with 50, 100, and 200 ME μg/ml reduced the size of BxPC-3 spheroids (Fig. 7A). With these changes in sphere size, ME treatment (50, 100, and 200 μg/ml) for 12 days triggered a more compact and regular shape of BxPC-3 spheroidal cells by reducing their proliferation rate. While 100 and 200 μg/ml of ME treatment for three days only slightly inhibited the growth of BxPC-3, treatment for day 12 effectively reduced 20% and 25% of the spheroid area, respectively (Fig. 7B). Thus, these data suggest that ME could suppress the growth of 3D-cultured cancer spheroids and 2D cultured cells.

Discussion
Cancer cells continuously require many nutrients, including oxygen, glucose, amino acids, and lipids, for uninhibited proliferation [28,31]. Therefore, cancer cells alter their energy metabolism pathways to support the massive demand for nutrients by inducing anaerobic glycolysis and recycling intracellular organelles [31,32]. Although intracellular metabolic reprogramming is a general feature of cancers, each has a different metabolic alteration strategy suitable for its tissue, genetic mutation, or microenvironment [28,33]. In pancreatic cancer, metabolic reprogramming promotes cancer progression and metastasis and disrupts therapeutic efficacy [28]. Therefore, regulating metabolic pathways to block cancer progression is a promising therapeutic strategy for successfully managing pancreatic cancer. In this regard, we focused on the inhibitory proliferation effect of ME on BxPC-3 human pancreatic cancer cells via regulation of their energy metabolism.
The genus Mychonastes, a freshwater microalga, produces lipids and carotenoids [34,35]. Although recent studies have revealed the bioactivity of the extracts obtained from the Mychonastes genus, such as their antioxidant, anti-fungal, and anticancer effects, its intracellular pathway has not been elucidated [11,36].
Through GC-MS/MS analysis to investigate the chemical components in ME, we found that β-sitosterol tetra-O-acetyl-β-D-glycopyranoside (C 43 H 68 O 10 ) was the most abundant component of the ME (Fig. 1, Table 1). β- Sitosteryl is a phytosterol, a naturally occurring compound structurally like cholesterol [37]. Recent studies have investigated phytosterols' anticancer effects through cell cycle arrest, apoptosis induction, and blocking angiogenesis [38]. For example, Xu et al. revealed that β-Sitosterol-D-glucoside promoted apoptosis of breast cancer via microRNA-10a regulation of the PI3K-Akt pathway [38]. Furthermore, according to a study by Swadesh et al., β-Sitosterol-3-O-β-D-glucoside from Azadirachta indica triggered apoptosis via DNA fragmentation-mediated G0/G1 arrest in leukemic cells [39]. Moreover, stigmasta-3α,5α-diol 3-O-β-D-glucopyranoside and glycocholic acid, the next most abundant components in ME, were also related to steroids from plants and lipid emulsification [40]. Therefore, we can deduce that abundant β-sitosterol in ME may inhibit pancreatic cancer growth.
In addition to β-sitosterol, microalgal carotenoids or pigments, such as astaxanthin, beta-carotene, and phycoerythrin, have been studied for inducing cell cycle G0/G1 phase arrest in cancer cells [41,42]. Accordingly, even a small amount of various ME components might comprehensively inhibit cell cycle progression in BxPC-3 cells.
As shown in Fig. 4, ME treatment strikingly inhibited the migration of BxPC-3 cells. Cytoskeleton remodeling is necessary during cell cycle progression and migration [43]. The PI3K signaling pathway regulates cytoskeleton rearrangement, including actin polymerization, an abundance of intermediate filaments, and microtubule stability [44,45]. The reduced PI3K activation by ME treatment may suppress pancreatic cancer migration (Figs. 4-6). The migration-inhibitory effect of ME could increase its therapeutic efficacy, in hand with its growthinhibitory effect on pancreatic cancer cells.
On the pancreatic cancer surface, insulin-like growth factor (IGF) and IGF receptors are highly expressed to promote cancer progression [45]. Binding of IGF to the IGF receptor activates PI3K/Akt and ERK signaling leading to cancer cell survival, proliferation, and migration [46]. Conversely, IGFBP suppresses IGF-related signaling pathways by interrupting the IGF-to-IGF receptor binding [47,48]. In our study, we found that ME treatment upregulated the expression of IGFBP3, the most abundant IGFBP, and downregulated the activation of the PI3K-mTOR signaling pathway (Figs. 5 and 6). According to a study by Kerr et al.,, levels of which are increased by β-sitosterol, acts through the IGF-and IGFBP-related pathways in cancer [48]. This suggests that βsitosterol in ME may play a crucial role in modulating the IGFBP-PI3K pathway in pancreatic cancer cells. However, ME treatment induced Akt phosphorylation and inactivated PI3K and mTOR (Fig. 6B). Lakshmipathi et al. reported that Akt activation might affect the intracellular energy environment [49]. In a metabolic inhibitory situation, downregulated mTOR activates Akt through negative feedback [49]. Akt activation might be more sensitively induced because mTOR is highly activated in pancreatic cancer cells [17]. This suggests that IGFBP3-mediated growth-inhibitory signals may cause the upregulation of Akt phosphorylation in ME-treated cancer cells.
AMPK is a crucial regulator of intracellular energy homeostasis and interacts with the mTOR signaling [49]. AMPK activation regulates protein synthesis through mTOR signaling-mediated p70S6K and 4EBP1 modulation [50,51]. We also determined the expression of AMPK, a crucial pathway, and mTOR downstream effector of PI3K-Akt signaling based on STRING analysis. ME treatment triggered 4E-BP1 activation and mTOR inactivation, whereas the activation of AMPK and p70S6K was slightly altered (Fig. 6). According to Sook et al., β-sitosterol induces ROS-mediated AMPK and MAPK activation [52]. Therefore, the slight increase in AMPK activation by ME treatment might be due to the inactivation of PI3K and activation of Akt and β-sitosterol in ME (Fig. 6A). However, β-Sitosterol is also known as its antioxidant activity [53]. Thus, correlation ROS and ME is needed to be clarified. mTOR, a master regulator of energy homeostasis, plays a crucial role in coordinating cancer cell proliferation and growth [54,55]. In particular, the inactivation of mTOR can induce cell cycle G1 phase arrest [19,54]. 4E-BP1 and p70S6K, modulated by mTOR, are significant mediators of the mTOR-dependent G1 arrest [19,54]. As shown in Figs. 3 and 6, we confirmed that ME treatment markedly suppressed the activation of mTOR and 4E-BP1 with G1 phase arrest. In contrast to p70S6K, 4E-BP1 directly affects cell cycle regulation via the CAP-dependent mRNA translation [56]. This may correlate with the significant increase in 4E-BP1 activation in ME-treated BxPC-3 cells (Fig. 6B). The regulation of mTOR or AMPK by microalgal extracts has been reported previously, but this study is the first to show that ME treatment changed the 4E-BP1 and p70S6K activation [41,57,58].
Like ME, Spirulina microalgae extracts have also been reported to have growth-inhibitory effects on pancreatic cancer cells [59,60]. Phycocyanin, a pigment-protein in Spirulina extracts, functions as an anticancer compound that regulates PI3K/AKT/mTOR or AMPK signaling [59,61]. Thus, analyzing chemical components in microalgal extracts is essential to clarify the correlation between extracts and their effects. This study provides a critical clue for the anticancer effect of ME, from its chemical composition to the intracellular mechanism of human pancreatic cancer cells.
A two-dimensional culture system cannot represent the cellular microenvironment in our body; hence, a 3D cell culture model that is closer in vivo, with limited nutrient supply, polarity, and cell-cell interaction, is in the limelight in the fields of drug discovery and drug repositioning [62]. Therefore, it is crucial to investigate drug efficacy using 3D cultured cancer cells, as it can provide accurate data for in vivo trials [63]. As shown in Fig. 7, ME treatment reduced the growth and irregularity of 3D cultured BxPC-3 spheroids. Since the loosened shape of spheroids is caused by low expression of E-cadherin, downregulation of E-cadherin expression could trigger cancer malignancy, including invasion, migration, and proliferation [64,65]. Hence, ME treatment may hamper the progression of pancreatic tumors, as with 3D cultured cancer spheroids. These data indicate the high application potential of ME in the treatment of human pancreatic cancer.
In anticancer studies, reducing the side effects caused by killing normal cells is a major clinical issue [66]. In particular, rapidly growing normal cells such as bone marrow, germline, and hair papilla cells are vulnerable to chemotherapeutic agents [67]. Our study confirmed that ME treatment was not highly toxic to bone marrowderived hMSC (Fig. 2). Furthermore, since mTOR levels are markedly higher in pancreatic cancer cells than in normal cells, ME may sensitively act on pancreatic cancer cells than on normal cells. These results show that ME is a prospective anticancer agent with few side effects.
Our study clarified that ME treatment increased cell cycle G1 phase arrest in pancreatic cancer cells for the first time by disrupting energy homeostasis via the IGFBP-PI3K-mTOR signaling pathway (Fig. 8). Additionally, our findings suggest that ME treatment could block the growth of both 2D and 3D cultured pancreatic cancer cells without toxicity to normal cells. In summary, ME represents a potential biosource for anticancer drug development.